Skeletal muscles produce reactive oxygen species (ROS) in response to a variety of stress stimuli, including thermal stress, osmotic stress, intense stimulation and hypoxia. These signals appear to be functionally significant but do not cause injury or damage under most normal physiologic conditions. We hypothesize that ROS, in this setting, play important roles in signaling networks designed to assist cells to withstand stress. The focus of the current proposal will be on the ROS produced in the transition from high to low O2 in skeletal muscle. This phenomenon is coincident with the hypoxia-induced shift in the redox state of the cell (NADH/NAD+), but we do not know if the signal arises from changes in redox or some other hypoxia- induced cellular response. We also do not know the sub-cellular origins of this signal or what phenotype produces it. AIM 1 of the proposal will identify the primary cellular and subcellular origins of ROS formation produced during metabolic stress and it will determine the sensitivity of the ROS-generating system to changes in PO2 vs. shifts in NADH/NAD+. To address this aim we have designed new imaging methods, including multiphoton and fluorescence lifetime, and a new fluorescent probe for localization of superoxide close to membranes. We will also determine the critical stimulus modality and intensity by measuring, and independently manipulating PO2 and cell redox state. In AIM 2, we will study the functional significance of hypoxia-induced ROS. We hypothesize that stress-induced ROS promotes energy mobilization and inhibits energy expenditure. First, we will evaluate the potential role of AMP-dependent protein kinase and glycolytic flux as a possible target for stress-induced ROS. Second, we will determine how ROS influences the relationships between Ca+2 release and force and the potential roles ROS and cell redox state have in altering Ca+2-induced force. This basic science investigation will give new insights into fundamental skeletal muscle biology that will have applications to a variety of muscle disorders related to O2 transport limitation.